The objective of this project is to gain a better understanding of the mechanisms which support the function of the cholinergic synapse. Experiments will be designed and carried out to determine if the high affinity choline co-transporter is found in two distinct populations in cholinergic terminals in the horseshoe crab. It is anticipated that one population exists in the terminal membrane and a second exists in cytoplasmic vesicles in the terminal. Efforts will be made to identify, separate, and characterize these two populations of choline co- transporters. Successful completion of this project will have significant implications for studying the molecular mechanisms undergirding membrane trafficking of the choline co-transporter. The following specific aims are set forth: 1. To demonstrate that hemicholinium -3(HC-3) binding is associated selectively with cholinergic terminals and that HC-3 binding is localized in the presynaptic membrane of ChAT identified terminals; (initiated) 2. To demonstrate that elevated K+ stimulated increase in choline uptake is accomplished by a quantifiable increase in HC-3 binding in cholinergic terminals; (completed) 3. To demonstrate that ACh vesicles and choline co-transporter vesicles are different populations; to isolate separable populations of synaptic and synaptic-like vesicles - One enriched with the VAChT and ACh; the other enriched with HC-3 binding sites; (initiated) 4. To demonstrated physiological characteristics of the Ch co-transporter in the isolated synaptic-like vesicle population (fraction)enriched with HC-3 binding; and 5. To demonstrate PKC localization in cholinergic neurons and terminals. To determine if one specific isoform or multiple isoforms of PKC are localized in cholinergic terminals. (initiated)